Sustained release formulations using non-aqueous carriers

ABSTRACT

The disclosure provides one-component, injectable, sustained release formulations which comprise microspheres containing active pharmaceutical ingredients (e.g., exenatide), wherein the microspheres are suspended in a non-aqueous carrier. The non-aqueous carrier can be an oil, a fractionated oil, triglycerides, diglycerides, monoglycerides, propylene glycol fatty acid diesters, and the like. The formulations offer distinct advantages of long shelf life for the stability and potency of the formulation and sustained release of active pharmaceutical ingredients to reduce the frequency of medication dosing and to increase patient compliance.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/898,381, filed Feb. 16, 2018, which is a continuation of U.S. patentapplication Ser. No. 15/234,021, filed Aug. 11, 2016, which is acontinuation of U.S. application Ser. No. 14/524,521, filed Oct. 27,2014, which is a continuation of U.S. application Ser. No. 13/060,225,filed May 16, 2011 and issued as U.S. Pat. No. 8,895,033, which is aNational Stage Entry of International Application No. PCT/US2009/056058,filed Sep. 4, 2009, which claims priority to U.S. ProvisionalApplication No. 61/094,381 filed on Sep. 4, 2008; all of which areincorporated herein by reference in their entireties.

BACKGROUND

Injectable sustained release formulations offer the opportunity toprovide therapeutic amounts of active pharmaceutical ingredients over anextended period of time from a single injection, thus eliminating theneed for once or twice daily injections. Presently available injectablesustained release formulations utilizing, for example, microspheres andan aqueous carrier, carry several disadvantages. The formulations do notoffer long term stability in the aqueous carrier, thus necessitatingseparate packaging and storage for the microspheres and aqueous carrier,and the patient must take several steps to combine the microspheres andaqueous carrier before administering the injection.

Another disadvantage of presently available injectable microsphereformulations is a large burst release following injection, which causesan undesirable in vivo release of active pharmaceutical ingredient in asingle burst. When medications have toxic or deleterious side effects,this is undesirable.

There is a need for formulations and methods of safely administeringsustained release pharmaceutical formulations to patients so that theactive ingredient will be released in vivo over an extended period oftime and without an unacceptable initial burst release. Ideally theactive ingredient is released so as to maintain levels within thetherapeutic window, i.e., in the concentration range above that neededto cause the desired clinical effect, but below that where undesirableside effects outweigh the benefits of the drug. It is also necessarythat this active pharmaceutical ingredient be provided in a manner thatis easy and convenient for the patient to self-administer and that isprovided in a formulation that maintains stability for a long period oftime in a liquid state. The disclosure is directed to these as well asother important ends.

SUMMARY

The disclosure provides formulations comprising microspheres thatcontain active pharmaceutical ingredients, where the microspheres aresuspended in a non-aqueous pharmaceutically acceptable carrier. Theformulations are one-component injectable microsphere formulations, suchthat they do not require the patient to mix the formulation with apharmaceutically acceptable carrier prior to injection. The disclosureoffers distinct advantages over prior two-component formulations byproviding for a long shelf life of the composition in the carrier,sustained release of the active pharmaceutical ingredient, a lesscomplex carrier, a more easily manufactured carrier, a less complexinjection-delivery apparatus, a kit with less components, and ease ofuse by patients.

The disclosure provides sustained release formulations comprising apharmaceutically acceptable carrier which consists essentially of one ormore triglycerides which comprise C₆-C₁₂ fatty acids; and microsphereswhich consist essentially of a poly(lactide-co-glycolide) polymer havingdispersed therein about 1% to 10% (w/w) exenatide and about 0.1% to 5%(w/w) of a sugar; wherein the ratio of lactide:glycolide in the polymeris about 70:30 to 30:70, or about 1:1. In one embodiment, the exenatideis present in an amount of 1% to 5% (w/w) or 5% (w/w) and the sugar ispresent in an amount of 2% (w/w). The sugar may be, e.g., glucose,dextrose, galactose, maltose, fructose, mannose, sucrose, lactose,trehalose, raffinose, acarbose, glycol, glycerol, erythritol, threitol,arabitol, ribitol, sorbitol, dulcitol, iditol, isomalt, maltitol,lactitol, mannitol, xylitol, or a combination of two or more thereof. Inone embodiment, the sugar is sucrose. The formulation is a suspensionwhereby the microspheres are suspended in the carrier. In oneembodiment, the total pore volume of the microspheres is about 0.1 mL/gor less, as determined using mercury intrusion porosimetry, to provide arelease profile having a ratio of maximum serum concentration ofexenatide during the period of release (C_(max)) to average serumconcentration of exenatide during the period of release (C_(ave)) ofabout 3 or less. Further, although the microspheres are formulated inoil (i.e. a carrier as disclosed herein), the microspheres do notnecessarily have oil contained within the interior spaces or pores, orwithin a substantial number of interior spaces or pores, of themicrospheres, and yet can achieve the surprising properties disclosedherein.

The disclosure provides sustained release formulations comprising apharmaceutically acceptable non-aqueous carrier and microspheres whichcomprise a biocompatible, biodegradable polymer and an activepharmaceutical ingredient. In one embodiment, the total pore volume ofthe microspheres is about 0.1 mL/g or less, as determined using mercuryintrusion porosimetry, to provide a release profile having a ratio ofmaximum serum concentration of the active pharmaceutical ingredientduring the period of release (C_(max)) to average serum concentration ofthe active pharmaceutical ingredient during the period of release(C_(ave)) of about 3 or less. Further, although the microspheres areformulated in oil (i.e. a carrier as disclosed herein), in someembodiments the microspheres do not have oil contained within theinterior spaces or pores, or do not have oil within a substantial numberof interior spaces or pores of the microspheres, and yet can achieve thesurprising properties disclosed herein. The formulation is a suspensionwhereby the microspheres are suspended in the carrier. The non-aqueouscarrier may be an oil, such as fractionated oils, triglycerides,diglycerides, monoglycerides, propylene glycol fatty acid diesters, andthe like.

In one embodiment the active ingredient is not soluble in the carrier.In various other embodiments the active ingredient has a solubility inthe carrier of less than 0.01 mg/ml, or less than 0.05 mg/ml or lessthan 0.1 mg/ml or less than 0.5 mg/ml or less than 1 mg/ml. In stillother embodiments the active pharmaceutical ingredient has a solubilityin the carrier such that less than 10% of the active ingredient in theformulation is contained within the carrier with the remaining 90%contained within the microparticles. In further embodiments less than 5%or less than 2% or less than 1% or less than 0.5% of the activeingredient is contained in the carrier. In still further embodimentswhere it is desirable to have some active ingredient immediatelyavailable, it may also be directly incorporated into the carrier in apharmaceutically effective amount.

The disclosure provides a kit, available to a patient or medical serviceprovider. The kit contains a container having a formulation of theinvention, and instructions for use. In one embodiment the container isa pen injector. The pen injector can be a single-dose pen injector or amulti-dose pen injector. In one embodiment the container is a vial,which can be either a single-dose vial or a multi-dose vial. In anotherembodiment the container is a cartridge, such as a cartridge for use ina injection apparatus. The cartridge can be either a single-dose or amulti-dose cartridge. In different embodiments the kit contains 1, 2, 3,4, or even 5 or more such containers carrying a formulation of theinvention. One further advantage of the formulations is that in oneembodiment the container is provided preservative free. But in otherembodiments a preservative can be soluble in the selected carrier andprovided in the formulation.

BRIEF DESCRIPTION OF THE DRAWINGS

For each of FIGS. 1-6, the microspheres comprise apoly(lactide-co-glycolide) copolymer having exenatide dispersed therein,as described in Example 1. For each of FIGS. 2-6, the oil carrier is amedium chain triglyceride (MCT) commercially available as MIGLYOL® 812(Sasol Germany GmbH, Witten, Germany).

FIG. 1 provides a comparison of the pharmacokinetics of four differentformulations of microspheres. In three formulations, the carrier was anoil (e.g., sesame oil; MIGLYOL® 812; ethyl oleate). In the comparativeformulation, the carrier was an aqueous diluent.

FIG. 2 is a graphical simulation (i.e., nanoparametric superposition) ofdata extrapolated from FIG. 1 of the plasma exenatide concentration overtime for the microsphere formulation comprising the oil carrier and themicrosphere formulation comprising the aqueous carrier in male SpragueDawley Rats. The plasma concentration plateau of exenatide may bereached after about 5 dosings.

FIG. 3 illustrates the in vitro release for a formulation comprisingmicrospheres in an oil carrier compared to formulations comprisingmicrospheres in an aqueous carrier.

FIG. 4 illustrates the in vivo release profile in rats over 10 hours fora formulation comprising microspheres in an oil carrier and aformulation comprising microspheres in an aqueous carrier.

FIGS. 5A and B illustrate the purity of exenatide over 9 months attemperatures of 5° C. and 6 months at 25° C. when stored in theformulations comprising the microspheres of Example 1 with an oilcarrier as compared to the purity of exenatide that was stored in drymicrospheres of Example 1. In FIG. 5A, the purity of exenatide wasdetermined by strong cation exchange HPLC. In FIG. 5B, the purity ofexenatide was determined by reverse-phase HPLC.

FIG. 6 illustrates the stability/potency of exenatide in a formulationwhere the microspheres are suspended in an oil carrier, where oneformulation is stored at 5° C. and one formulation is stored at 25° C.

DETAILED DESCRIPTION

The disclosure provides sustained release compositions provided inpharmaceutically acceptable carriers, for the sustained release of anactive pharmaceutical ingredient (API). The formulations may comprisemicrospheres comprised of a biocompatible, biodegradable polymer havingan active pharmaceutical ingredient dispersed therein, where themicrospheres are suspended in a non-aqueous carrier. The formulationsare one-component injectable formulations, compared to two-componentformulations which require that the microspheres be stored dry in onecontainer while the liquid carrier can be stored in a separatecontainer, such that the patient must mix the two together prior toinjection. The formulations offer the convenience of long term stabilityof a pharmaceutical composition in a non-aqueous liquid carrier, thuseliminating any need for the patient to add a pharmaceuticallyacceptable carrier to the pharmaceutical composition prior to injection.The formulations are provided in a single container for easy use by thepatient, who only need to lightly agitate the formulation beforeinjecting it from the same container. When the container provided isalso an injection device, even the step of syringing the formulation iseliminated. The formulations described herein offer the additionalimportant advantage of substantially reducing burst release of theactive pharmaceutical ingredient. Thus, even active pharmaceuticalingredients that have a toxic effect at higher concentrations can besafely administered using the formulations described herein.

The term “patient” refers to mammals, including humans, animal pets,farm animals, zoo animals, and the like. In one embodiment, the patientis a human.

The terms “treating” or “treatment” refer to the administration of oneor more active pharmaceutical ingredients to a patient who has acondition or disorder or a predisposition toward a condition ordisorder, with the purpose to alleviate, relieve, remedy, ameliorate,improve, slow or stop the progression or worsening of the disease, or atleast one symptom of the disease, condition or disorder, or thepredisposition toward the condition or disorder.

“Exenatide” has the same meaning and amino acid sequence as exendin-4.More particularly, exenatide is a synthetic peptide with the same aminoacid sequence as exendin-4, which is a peptide isolated from the venomof the Gila monster.

One Component Formulation

Previous injectable formulations contained at least two components. Thefirst component may be dry microspheres and the second component may bean aqueous pharmaceutically acceptable carrier. The first component andsecond component are stored in separate sealed containers (e.g., vials,injection pen chambers). The patient receives the two-componentformulation, and the patient or pharmacist must physically mix the twocomponents together prior to injection. In the case of an injection pen,the two components are mixed together immediately prior to injectioninto the patient. Two-component formulations typically are administeredto the patient within a short time after being mixed with thepharmaceutically acceptable carrier. For example, the microspherecomponent and the pharmaceutically acceptable aqueous carrier are mixedtogether and then the formulation is administered to the patient withinabout 30 or 60 minutes.

The formulations described herein are one component injectableformulations. A one component injectable formulation refers to aformulation that contains both the microspheres and the pharmaceuticallyacceptable carrier provided in the same container, and that may beadministered to the patient without the need to first combine themicrospheres and the pharmaceutically acceptable carrier. Accordingly,the one component formulation is manufactured as a pre-mixed formulationfor injection. A one-component formulation provides significantconvenience for manufacturing, transport, storage, and patient use.

In another embodiment the one-component formulation described herein isprovided in a sealed container. A “sealed container” is a container thathas not been opened, punctured, or had anything introduced into it sinceits time of completion of manufacture. The time of completion ofmanufacture is the time when the container holding the formulation isinitially sealed. Containers may include vials (single use ormulti-use), syringes, injection pens (e.g., single use or multi-use),and the like.

Carrier

“Carrier” (or vehicle) refers to a pharmaceutically acceptablenon-aqueous liquid material. The carrier is substantially inert so thatit does not interact with the microspheres described herein and isnon-toxic so that it does not negatively impact the patient. The carrieris preferably approved by or is awaiting approval by a regulatory agencyof the Federal or a state government or listed in the U.S. Pharmacopoeiaor other generally recognized pharmacopoeia for use in mammals, such ashumans. The term “carrier” may include one or more compounds. Thecarrier is a non-solubilizing carrier, in that the carrier does notsolubilize the polymer(s) that forms the microspheres. In a furtherembodiment, the carrier does not solubilize the active pharmaceuticalingredient(s) within the microspheres. For example, the carrier will notsolubilize exenatide or other water-soluble therapeutic peptides orproteins.

The term “non-aqueous” does not exclude trace amounts of residual waterthat do not have a demonstrated negative impact on the stability of thesustained release compositions. Thus, a composition may have about 0.1%(w/v) water or even about 0.25% water or less than 0.1% (w/v) water orless than 0.25% (w/v) water and still be considered non-aqueous. Thecarrier does not solubilize the microspheres to the extent of having ademonstrated negative impact on the stability of the microspheres ordemonstrated loss of burst release control. In one embodiment, thecarrier does not enter or permeate the biocompatible, biodegradablepolymer and is not dispersed within the biocompatible, biodegradablepolymer. The carrier also does not cause swelling of the microspheres toan extent that has a demonstrated negative impact on the stability ofthe microspheres. For example swelling may occur to a degree of lessthan 1% and still be considered a non-aqueous carrier that isnon-swelling of the microspheres.

In one embodiment, the non-aqueous carrier is a pharmaceuticallyacceptable oil. An oil is a substance that is in a viscous liquid stateat ambient temperatures or slightly warmer, and is both hydrophobic(immiscible with water) and lipophilic (miscible with other oils,literally). Exemplary pharmaceutically acceptable oil carriers includevegetable oils and volatile essential oils. Exemplary pharmaceuticallyacceptable oil carriers include coconut oil, palm oil, palm kernel oil,sesame oil, soybean oil, almond oil, rapeseed oil, corn oil, sunfloweroil, peanut oil, olive oil, castor oil, soybean oil, safflower oil,cottonseed oil, ethyl oleate, and the like. The carrier may comprise oneoil or a combination of two or more oils.

In one embodiment, the carrier is a fractionated oil or a combination oftwo or more fractionated oils. Exemplary pharmaceutically acceptable oilcarriers include fractionated coconut oil, fractionated palm oil,fractionated palm kernel oil, fractionated sesame oil, fractionatedsoybean oil, fractionated almond oil, fractionated rapeseed oil,fractionated corn oil, fractionated sunflower oil, fractionated peanutoil, fractionated olive oil, fractionated castor oil, fractionatedsoybean oil, fractionated safflower oil, fractionated cottonseed oil,and the like. In one embodiment, the carrier is fractionated coconutoil. In one embodiment, the carrier is fractionated palm kernel oil. Inone embodiment, the carrier is a combination of fractionated coconut oiland fractionated palm kernel oil.

As used herein, fractionation is a process whereby long chain fattyacids are removed from the oil, such that the resulting fractionated oilsubstantially comprises medium chain triglycerides. The skilled artisanwill appreciate that some long-chain fatty acids may remain in thefractionated oil, but generally in amounts less than 5 wt % or less than2 wt % of the total fatty acid content of the fractionated oil.

In one embodiment, the carrier is a long chain triglyceride, a mediumchain triglyceride, a diglyceride, a monoglyceride, a propylene glycolfatty acid diester, or a combination of two or more thereof.

In one embodiment, the carrier is a medium chain triglyceride. Themedium chain triglyceride may be synthetic or natural (e.g., producedfrom fractionated oils, such as coconut oil and/or palm kernel oil).“Medium chain triglyceride” refers to esters of glycerol having three C₆to C₁₂ fatty acid chains, where the three fatty acid chains may be thesame or different. Medium chain triglycerides are represented by thecompound of Formula (I):

wherein each x is independently 4, 6, 8, or 10. When x is 4, the chainis referred to as a C₆ fatty acid. When x is 6, the chain is referred toas a C₈ fatty acid. When x is 8, the chain is referred to as a C₁₀ fattyacid. When x is 10, the chain is referred to as a C₁₂ fatty acid. Invarious embodiments, each x is the same integer; two x are the sameinteger and one x is a different integer; or each x is a differentinteger.

In various embodiment, the medium chain triglyceride comprises esters of(i) three C₈ fatty acids; (ii) three C₁₀ fatty acids; (iii) two C₈ fattyacids and one C₁₀ fatty acid; (iv) two C₁₀ fatty acids and one C₈ fattyacid; (v) two C₈ fatty acids and one C₆ fatty acid; (vi) two C₁₀ fattyacids and one C₆ fatty acid; (vii) one C₈ fatty acid, one C₁₀ fattyacid, and one C₆ fatty acid; or (viii) any other combination of C₆, C₈,C₁₀, and C₁₂ fatty acids. In one embodiment, the medium chaintriglyceride comprises two C₈ fatty acids and one C₁₀ fatty acid. In oneembodiment, the medium chain triglyceride comprises two C₁₀ fatty acidsand one C₈ fatty acid.

The skilled artisan will appreciate that a mixture of medium chaintriglycerides may result from any process (e.g., fractionation,hydrogenation) used to prepare medium chain triglycerides. For example,substantially all of the medium chain triglycerides obtained fromfractionated coconut oil may comprise C₈ and/or C₁₀ fatty acids;however, there may be some medium chain triglycerides containing C₆and/or C₁₂ fatty acids.

In one embodiment, the medium chain triglycerides comprise esters of (i)0 to 2 wt % C₆ fatty acid, 65 to 80 wt % C₈ fatty acid, 20 to 35 wt %C₁₀ fatty acid, and 0 to 2 wt % C₁₂ fatty acid; (ii) 0 to 2 wt % C₆fatty acid, 50 to 65 wt % C₈ fatty acid, 30 to 45 wt % C₁₀ fatty acid,and 0 to 2 wt % C₁₂ fatty acid; (iii) 0 to 2 wt % C₆ fatty acid, 45 to65 wt % C₈ fatty acid, 30 to 45 wt % C₁₀ fatty acid, 0 to 3 wt % C₁₂fatty acid; and 0 to 5 wt % linoleic acid; or (iv) 0 to 2 wt % C₆ fattyacid, 45 to 55 wt % C₈ fatty acid, 30 to 40 wt % C₁₀ fatty acid, 0 to 3wt % C₁₂ fatty acid, and 10 to 20 succinic. In one embodiment, themedium chain triglyceride comprises 0 to 2 wt % C₆ fatty acid, 50 to 65wt % C₈ fatty acid, 30 to 45 wt % C₁₀ fatty acid, and 0 to 2 wt % C₁₂fatty acid, and which is commercially available as MIGLYOL® 812 (SasolGermany GmbH, Witten, Germany) The weight % is based of the total fattyacid content of the triglycerides. In one embodiment, the medium chaintriglycerides may comprise up to 2% C₁₄ fatty acids.

The carrier may comprise one, two, three, four or more different mediumchain triglycerides. In one embodiment, the carrier comprises a mediumchain triglyceride comprising esters of two C₈ fatty acids and one C₁₀fatty acid. In one embodiment, the carrier comprises a medium chaintriglyceride comprising esters of one C₈ fatty acid and two C₁₀ fattyacids. In one embodiment, the carrier comprises two different mediumchain triglycerides, where a first medium chain triglyceride comprisesesters of two C₈ fatty acids and one C₁₀ fatty acid and a second mediumchain triglyceride comprises esters of one C₈ fatty acid and two C₁₀fatty acids. In one embodiment, the carrier comprises a medium chaintriglyceride which comprises 0 to 2 wt % C₆ fatty acid, 50 to 65 wt % C₈fatty acid, 30 to 45 wt % C₁₀ fatty acid, 0 to 2 wt % C₁₂ fatty acid,based on the total fatty acid content of the medium chain triglyceride.

The triglycerides may be prepared by methods known in the art and arecommercially available as MIGLYOL® 810, 812, 818, 829 (Sasol GermanyGmbH, Witten, Germany) or NEOBEE® 1053, 895, M-5 (Stepan Company,Northfield, Ill.).

In another embodiment the carrier is a propylene glycol diester ofsaturated vegetable fatty acids with chain lengths of C₈ and C₁₀(caprylic and capric acid). An example of one such commerciallyavailable carrier is MIGLYOL® 840 (Sasol Germany GmbH, Witten, Germany).The pharmaceutically acceptable, non-aqueous carrier may optionallycomprise other pharmaceutically acceptable excipients. Exemplaryexcipients include sugars (e.g., sucrose, glucose, dextrose, galactose,maltose, trehalose, fructose, maltodextrin); sugar alcohols (e.g.,glycol, glycerol, erythritol, threitol, arabitol, ribitol, sorbitol,dulcitol, iditol, isomalt, maltitol, lactitol, mannitol, xylitol);preservatives (e.g., benzoic acid, sorbic acid, meta cresol, sodiumbenzoate, potassium sorbate, methylparaben, propylparaben, butylparaben,benzalkonium chloride, and the like, generally oil-soluble, with somesolubility in the selected carrier); and antioxidants (e.g., sodiummetabisulfite, butylated hydroxy anisole, butylated hydroxy toluene,sodium sulfite, tocopherol, thymol, ascorbate, propyl gallate, and thelike). In one embodiment, the carrier optionally comprises mannitol,maltodextrin, sorbitol, or a combination of two or more thereof.

The pharmaceutically acceptable carrier may contain a gel-forming agent;however, the gel-forming agent may only be present in an amount thatdoes not cause a gel-depot to form at the site of in vivo administrationof the formulation. In one embodiment, the pharmaceutically acceptablecarrier does not contain a gel-forming agent. Exemplary gel-formingagents include cellulose derivatives (e.g., hydroxypropyl cellulose,carboxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl methylcellulose, methylcellulose); polyoxyethylene and polyoxypropylenepolymers or co-polymers (poloxamers); chitosan acid, and the like. Theskilled artisan will understand that the formation of gels in vivo canbe determined by methods known in the art, such as the use ofhistological sections and colored dyes.

In certain embodiments the non-aqueous, non-solubilizing carrier has aviscosity of from 5 cP to 200 cP or from 10 cP to 90 cP. In otherembodiments the viscosity of the non-aqueous, non-solubilizing carrieris from 20 cP to 80 cP or from 30 cP to 70 cP. Thus, with reference tothis disclosure the person of ordinary skill will be able to identifyother oils, triglycerides, or non-aqueous compounds that also can bepresent in the non-aqueous, non-solubilizing carrier.

Microspheres

The term “microspheres” includes microspheres, microparticles,nanoparticles, pellets, cylinders, rods, discs, and the like. Amicrosphere can have a spherical, non-spherical or irregular shape. Themicrosphere will be of a size suitable for injection. A typical sizerange for microspheres is 1000 microns or less. In a particularembodiment, the microsphere ranges from about one to about 180 micronsin diameter. In yet further embodiments suitable release profiles areobtained when microspheres range from about 1 to 100 microns, from about30 to 90 microns, or from about 50 to 70 microns. In one embodiment themean microsphere size is not less than or is equal to about 50, 60 or 70microns, and preferably less than about 80, 90, or 100 microns. Atlarger sizes, microsphere are preferably substantially non-aggregated toallow passage through a 25 gauge needle, or a 27 gauge needle, or a 30gauge needle, or a 31 gauge needle.

Consistent and superior release profiles are obtained by controllingsize distribution. In one embodiment a mean microsphere size is about 50microns and the lower and upper range of microsphere are about 30 and 90microns, respectively. Distribution of microspheres can be describedusing a mean diameter of the volume. Mean diameter of the volumedistribution represents the center of gravity of the distribution and isa type of “average particle size.” In various embodiments, themicrospheres have a mean diameter of the volume distribution of about 50to 70 microns, about 50 to 60 microns or about 50, 60 or 70 microns,with a Distribution of Volume (DV) of less than or about 5%, 10%, or 15%at 30 microns and a DV of greater than or about 80%, 85%, 90% or 95% at90 microns. In one embodiment, the microspheres have a mean diameter ofthe volume distribution of about 60 microns, with a Distribution ofVolume (DV) of less than or about 10% at 30 microns and a DV of greaterthan or about 90% at 90 microns.

Microspheres may be prepared by processes known in the art anddescribed, e.g., in U.S. Pat. Nos. 7,563,871, 7,456,254, 7,223,440,6,824,822, 6,667,061, 6,495,164, and 6,479,065, the disclosures of whichare incorporated by reference herein.

In a further embodiment, the microspheres have a less porous outerlayer, and further can have a non-porous outer layer. Accordingly, inthe formulations disclosed herein the oil does not have access to theinterior spaces or pores or even to a substantial portion of theinterior spaces or pores. It is specifically, contemplated that for eachof the formulations disclosed herein the microspheres can additionallylack oil (or a carrier as disclosed herein) in the interior spaces ofthe microspheres. Thus, the advantages of the present formulations canbe achieved without the presence of oil in the interior spaces of themicrospheres when formulated.

Polymers

The microspheres comprise biocompatible, biodegradable polymers. Apolymer is biocompatible if the polymer and any degradation products ofthe polymer are non-toxic to the patient at administered levels and alsopossess no demonstrated deleterious or untoward effects on the patient'sbody, for example a substantial immunological reaction at the injectionsite. Biodegradable means the polymer will degrade or erode in vivo toform smaller units or chemical species. Degradation can result, forexample, by enzymatic, chemical and physical processes.

Exemplary biocompatible, biodegradable polymers include, for example,polylactides, polyglycolides, poly(lactide-co-glycolides), polylacticacids, polyglycolic acids, poly(lactic acid-co-glycolic acid)s,polycaprolactones, polycarbonates, polyesteramides, polyanhydrides,polyamino acids, polyorthoesters, polycyanoacrylates, poly(p-dioxanone),polyalkylene oxalates, biodegradable polyurethanes, blends thereof andcopolymers thereof. Acceptable molecular weights for the biocompatible,biodegradable polymers can be determined by a person of ordinary skillin the art taking into consideration factors such as the desired polymerdegradation rate, physical properties such as mechanical strength, endgroup chemistry and rate of dissolution of polymer. Typically, anacceptable range of molecular weight is of about 2,000 Daltons to about2,000,000 Daltons. The biocompatible, biodegradable polymer can also beselected based upon the polymer's inherent viscosity. Suitable inherentviscosities are about 0.06 to 1.0 dL/g; about 0.2 to 0.6 dL/g; or about0.3 to 0.5 dL/g.

In one embodiment, the biocompatible, biodegradable polymer is apoly(lactide-co-glycolide) copolymer (also referred to as “PLGA”) havinga lactide:glycolide ratio from 70:30 to 30:70, or from 60:40 to 40:60 orabout 50:50. The molecular weight of the poly(lactide-co-glycolide)copolymer is about 10,000 Daltons to about 90,000 Daltons. In anotherembodiment, the molecular weight of the poly(lactide-co-glycolide)copolymer is about 30,000 Daltons to about 70,000, or from about 50,000to about 60,000 Daltons.

The formulation may contain microspheres at a concentration of from 1mg/ml to 500 mg/ml; from 25 mg/ml to 300 mg/ml; or from 50 mg/ml to 200mg/ml.

Active Pharmaceutical Ingredient

An active pharmaceutical ingredient is a biologically active compoundthat has a therapeutic, prophylactic, or other beneficialpharmacological and/or physiological effect on the patient. The activepharmaceutical ingredient can also be a mixture of two or morecompounds. The term “peptide” refers to any compound having two or moreconsecutive amino acids. As used herein, the term “peptide” issynonymous with peptide, polypeptide, and protein. In one embodiment,the peptide has a molecular weight of from 500 Da to 100 kDa; from 1 kDato 80 kDa; from 1 kDa to 50 kDa; from 1 kDa to 30 kDa; or from 1 kDa to20 kDa. In one embodiment, the peptide comprises 2 to 500 amino acidresidues; 2 to 250 amino acid residues; 5 to 100 amino acid residues; or5 to 50 amino acid residues.

In one embodiment, the active pharmaceutical ingredient is a GLP-1receptor agonist compound, such as an exendin, an exendin analog,GLP-1(7-37), a GLP-1(7-37) analog, and the like. Exemplary GLP-1receptor agonist compounds include exendin-3, exenatide, GLP-1(1-37),GLP-1(7-37)-NH₂, GLP-1(7-36), GLP-1(7-36)-NH₂, Leu¹⁴-exendin-4,Leu¹⁴,Phe²⁵-exendin-4, exendin-4(1-28), Leu¹⁴-exendin-4(1-28), Leu¹⁴,Phe²⁵-exendin-4(1-28), exendin-4(1-30), Leu¹⁴-exendin-4(1-30),Leu¹⁴,Phe²⁵-exendin-4(1-30), liraglutide, and the compounds describedin, e.g., U.S. Pat. Nos. 7,157,555, 7,220,721, 7,223,725, and WO2007/139941, the disclosures of which are incorporated herein byreference.

Other peptides known in the art can be used as the active pharmaceuticalingredient in the formulations described herein. Exemplary peptidesinclude amylin, amylin agonists (e.g., pramlintide, davalintide,Val²⁷-davalintide); leptin, leptin agonists (e.g., metreleptin);PYY(3-36) and agonist analogs thereof; glucagon, glucagon agonists,glucagon antagonists, peptide chimera of GLP-1 receptor agonists andglucagon agonists, peptide chimera of human amylin and salmoncalcitonin, insulin, heparin, low-molecular weight heparin, angiotensin,argipressin, argireline, atosiban, bivalirudin, cetrorelix,desmopressin, enfuvirtide, deptifibatide, GHRP-2, GHRP-6, gonadorelin,leuprolide, lysipressin, melanotan, nesiritide, octreotide, oxytocin,PT141, calcitonin, sermorelin, somatostatin, terlipressin, thymopentin,thymosin al, triptorelin, vapreotide, elcatonin, ziconotide, ghrelin,nafarelin, BNP-32, and the like.

The active pharmaceutical ingredient can also be a small molecule. A“small molecule” is an organic molecule. Exemplary small moleculesinclude metformin, sulfonylureas, TZDs, statins (e.g., atorvastatin,cerivastatin, fluvastatin, Lovastatin, mevastatin, pitavastatin,pravastatin, rosuvastatin, simvastatin); non-selective beta blockersand/or alpha-1 blockers (e.g., carvedilol, dilatrend, eucardic, carloc);PDE3 inhibitors (e.g., cilostazol); antiplatelet drugs, antithromboticdrugs, anticoagulant drugs, glycoprotein IIb/IIIa inhibitors (e.g.,abciximab, eptifibatide, tirofiban); antibacterial drugs (e.g.,ciprofloxacin, norfloxacin, levofloxacin, moxifloxacin, sparfloxacin,gemifloxacin, ecinofloxacin, delafloxacin); Factor Xa inhibitors (e.g.,glycosaminoglycans, oligosaccharides, heparinoid); direct Xa inhibitors(e.g., xabans); direct thrombin (II) inhibitors (e.g., hirudin,argatroban, dabigatran, melagatran, ximelagatran, defibrotide,ramatroban, antithrombin III, protein C); thrombolytic drugs (e.g.,plasminogen activators, urokinase, streptokinase, serineendopiptidases); ACE inhibitors (e.g., lisinopril, aceon, acertil,armix, coverene, coverex, coversum, prestarium, prexanil, Prexum,procaptan); ADP receptor/P2Y12 inhibitors (e.g., clopidogrel,ticlopidine, prasugrel); prostaglandin analogs (e.g., beraprost,prostacyclin, iloprost, treprostinil); anticoagulants (e.g., coumarin,coumatetralyl, dicoumarol, ethyl biscoumacetate, phenprocoumon,warfarin, clorindione, diphenadione, phenindione, tioclomarol);diuretics (e.g., hydrochlorothiazide); macrolides (e.g., azithromycin,clarithromycin, dirithromycin, erythromycin, roxithromycin,telithromycin); NSAIDs and COX-3 inhibitors (e.g., celecoxib,etoricoxib, parecoxib); sulphonanilides (e.g., nimesulide), and thelike.

The skilled artisan will appreciate that the formulations describedherein may contain two or more peptides; two or more small molecules; ora combination of small molecules and peptides. For example, theformulation may comprise two different sets of microspheres, where oneset of microspheres contain one peptide (e.g., pramlintide) and anotherset of microspheres contain a different peptide (e.g., metreleptin). Inone embodiment, 1 to 99% of the microspheres comprise one activepharmaceutical ingredient and 99 to 1% of the microspheres comprise adifferent active pharmaceutical ingredient. In another embodiment 30 to70% of the microspheres comprise one active pharmaceutical ingredientand 70 to 30% of the microspheres comprise a different activepharmaceutical ingredient. The skilled artisan will appreciate that thepercentage of each type of peptide in the formulation will be determinedby the relative potency of the peptides. This formulation advantageouslyallows high potency peptides to be combined with low potency peptidesfor simultaneous delivery to a patient because the low potency peptidescan be provided in more microspheres and the high potency peptides canbe provided in fewer microspheres in the same formulation. Exemplarycombinations of peptides and/or small molecules that can be administeredin different sets of microspheres and in the same formulation include:pramlintide and insulin; pramlintide and metreleptin; davalintide andmetreleptin; exenatide and metreleptin; lovastatin and niacin;atorvastatin and amlodipine; simvastatin and ezetimibe; exenatide andmetformin; and the like.

The formulations generally contain from about 0.01% (w/w) to about 50%(w/w) of the active pharmaceutical ingredient (based on the total weightof the composition). For example, the amount of active pharmaceuticalingredient can be from about 0.1% (w/w) to about 30% (w/w) of the totalweight of the composition. The amount of active pharmaceuticalingredient will vary depending upon the desired effect, potency of theagent, the planned release levels, and the time span over which thepeptide will be released. In certain embodiments, the range of loadingis between about 0.1% (w/w) to about 10% (w/w), for example, from 0.5%(w/w) to about 5% (w/w), or from 1% to 5% (w/w). When the activepharmaceutical ingredient is a GLP-1 receptor agonist, suitable releaseprofiles can be obtained when the active pharmaceutical ingredient, forexample exenatide, is loaded at about 2% w/w to about 7% w/w, includingat about 2% w/w/, about 3% w/w, about 4% w/w, about 5% w/w, about 6%w/w, or about 7% w/w.

Sugars

The microspheres may also comprise one or more sugars. A sugar is amonosaccharide, disaccharide or oligosaccharide or a derivative thereof.Sugar alcohols of monosaccharides are suitable derivatives of sugar.Monosaccharides include, but are not limited to, glucose, fructose andmannose. A disaccharide, as further defined herein, is a compound whichupon hydrolysis yields two molecules of a monosaccharide. Suitabledisaccharides include, but are not limited to, sucrose, lactose andtrehalose. Suitable oligosaccharides include, but are not limited to,raffinose and acarbose. The microspheres may further comprise glucose,dextrose, galactose, maltose, fructose, mannose, sucrose, lactose,trehalose, raffinose, acarbose, glycol, glycerol, erythritol, threitol,arabitol, ribitol, sorbitol, dulcitol, iditol, isomalt, maltitol,lactitol, mannitol, xylitol, or a combination of two or more thereof. Inone embodiment, the sugar is sucrose, glucose, mannose, or fructose. Inone embodiment, the sugar is sucrose.

The amount of sugar present in the microspheres can range from about0.01% (w/w) to about 50% (w/w), such as from about 0.01% (w/w) to about10% (w/w), such as from about 0.1% (w/w) to about 5% (w/w) of the totalweight of the composition. In one embodiment, about 2% (w/w) sucrose isused.

Alternatively, the amount of sugar present in the microspheres can bereferred to on a weight ratio with the active pharmaceutical ingredient.For example, the active pharmaceutical ingredient and sugar can bepresent in a ratio from about 10:1 to about 1:10 weight:weight. Inparticularly preferred embodiments, the ratio of active pharmaceuticalingredient (e.g., exenatide) to sugar (e.g., sucrose) is about 3:2(w/w), 4:2 (w/w), or 5:2 (w/w). Combinations of two or more sugars canalso be used. The amount of sugar, when a combination is employed, isthe same as the ranges recited above.

Sustained Release

The compositions are sustained release compositions, meaning that theactive pharmaceutical ingredient contained in the compositions will bereleased into the patient over an extended period of time such as, forexample, a period of two days, or three days, or at least two days, orat least three days, or over a period of one week, two weeks, one month,three months, or one year. The release of the active pharmaceuticalingredient is considered complete when there is no longer a therapeuticlevel of active pharmaceutical ingredient in the patient's body, asdetermined by the medical judgment of those of ordinary skill in theart.

Cmax as used herein is the maximum serum concentration of drug whichoccurs during the period of release which is monitored. Cave as usedherein, is the average serum concentration of drug derived by dividingthe area under the curve (AUC) of the release profile by the duration ofthe release.

In one embodiment the ratio of Cmax to Cave is about 3 or less. Thisprofile is particularly desirable for anti-diabetic or glucoregulatorypolypeptides, such as those described herein. A ratio of about 3 or lesscan provide a Cave in a therapeutic window while avoiding adverse drugside effects which can result from higher ratios. Further by controllingthe physical aspects of the sustained release composition, as describedherein, a superior desired release profile can be achieved andcontrolled, for example, by appropriate selection of carrier properties,such as viscosity. There is thus provided a reduced burst (i.e. initialrelease; e.g., Cmax at 0-1 day). In other embodiments the Cmax to Caveratio is from about 1 to about 3, or from 1 to 3, or from about 2 toabout 3, or from 2 to 3. Further, a Cmax, if present, can be shiftedfrom the burst or initial release period into the “sustained phase” ofrelease. In one embodiment the Cmax can occur at at least 7, 14, 21, 28,35 or 42 days post administration and can occur at any integer day inbetween. In a further embodiment the Cmax occurs at about 21 to 35 daysafter administration, and in yet another embodiment is at about 28 to 31days, and further at about 28 days after administration. In a furtherembodiment the maximal concentration of drug (e.g. plasma concentration)occurs at at least 7, 14, 21, 28, 35 or 42 days post administration andcan occur at any integer day in between. In yet a further embodiment themaximal concentration of drug occurs at about 21 to 35 days afteradministration, particularly in the case of glucoregulatory agents suchas exendin-4, GLP-1, GIP or their analogs.

Longer Shelf Life

One advantage offered by the present formulations is a longer shelf lifefor the formulation. It was discovered unexpectedly that sustainedrelease compositions retain remarkable stability when stored in anon-aqueous carrier as described herein. In one embodiment theformulation has a shelf life of at least 6 months. In other embodimentsthe formulation has a shelf life of at least 1 year, or at least 18months, or at least 2 years. By “shelf life” is meant the formulationcan be stored or maintained for that period of time under appropriateenvironmental conditions while retaining at least 90% of the desiredactivity of the active pharmaceutical ingredient relative to theactivity at initial formulation (as 100%). In another embodiment theactive pharmaceutical ingredient retains at least 95%, or at least 98%or at least 99% of its desired activity as compared to its activityimmediately before storage. When the formulation contains microspheres,shelf life also refers to the retention of particle size and/ormorphology of the microspheres. Retention of size morphology can bedetermined by microscopic examination, the use of which is known topersons of ordinary skill in the art.

When formulated as disclosed herein a peptide or protein as activeingredient is less susceptible to oxidation and to hydrolysis, eitherchemical or proteolytic, both during storage and during its sustainedrelease period after injection. The addition of an anti-oxidant or otherstabilizer is not required in these formulations, particularly thosewhere the carrier is a medium chain triglyceride.

Reduced Burst Release

Another advantage of the present formulations is that formulationsaccording to the present disclosure offer a significantly reduced burstrelease rate compared with other formulations. When previously availableinjectable sustained release formulations are injected into a patientthere is often a “burst” of active ingredient or agent associated withthe injection. Without wanting to be bound by any specific theory, it isbelieved this burst is caused by that quantity of active pharmaceuticalingredient in the formulation that is not retained within the polymerthat is released over time. By “burst release” is meant that quantity ofactive pharmaceutical ingredient released within the first 24 hoursafter injection. In other embodiments it is that quantity of active thatis release over 1 hour, or 2 hours, or 4 hours, or 8 hours, or 12 hoursafter injection. In various embodiments the formulation of the inventionhas a burst release after injection of less than 10% or less than 5%, orless than 3%, or less than 2.5%, or less than 2%, or less than 1% orless than 0.75% or less than 0.5% or less than 0.25% or less than 0.1%.Percentages refer to the percentage of the total amount of activepharmaceutical ingredient in the injected formulation. Followinginjection of the formulation in the patient, the burst release may occurat any time up to about 24 hours, thereafter there may be a lag timewhere substantially no active pharmaceutical ingredient is released fromthe microspheres, and then the polymeric microspheres begin degradingand releasing the active pharmaceutical ingredient. The skilled artisanwill appreciate that the time period when the burst release occurs mayvary from patient to patient.

Burst can be assessed by measuring the proportion of the total areaunder the curve for a particular time period following administration ofa drug. Area under the curve (AUC) is a well established measurement inthe pharmaceutical sciences and measures the amount of drug or activeingredient that reaches the bloodstream in a set period of time. As iswell known in the art, the period of time selected will varyingdepending on the time period the concentration of the drug in the bloodis expected to be detectable or within the drug's therapeutic window.AUC is calculated by plotting the concentration of the drug in theblood, for example plasma concentrations, at various times during theselected time period and then calculating the total area under the curveobtained. In one exemplary embodiment, the area under the curve ismeasured for a 42 day period and using the formulations describedherein, the release or burst as measured within the first 24 hours is 5%or less, 2% or less, 1.5% or less, 1% or less, or 0.5% or less of thetotal AUC. In another embodiment, the formulations described hereinresult in a burst or proportion of the AUC that is 20% or less, 15% orless, 10% or less, 5% or less, or 2% or less than that obtained when thesustained release composition is contained in a carrier in which theactive pharmaceutical ingredient is soluble.

In another embodiment, the formulations described herein limit initialburst such that the upper limit of the therapeutic window for the activepharmaceutical ingredient is not exceeded. The therapeutic window is therange of concentration of active pharmaceutical ingredient in thecirculation, above which the active pharmaceutical ingredient has itsdesired effect, but below the concentration at which the adverse effectsassociated with the active pharmaceutical ingredient outweigh thebenefits as would be generally accepted among physicians. In oneexemplary embodiment, the active pharmaceutical ingredient is anexendin, for example exenatide, or agonist analogue thereof, andadministration of the formulations described do not result in acirculating level of active pharmaceutical ingredient exceeding 400pg/ml during the first 24 hours following administration. In anotherexemplary embodiment the active pharmaceutical ingredient is an exendin,for example exenatide, or agonist analogue thereof, and administrationof the formulations described does not result in a circulating level ofactive pharmaceutical ingredient exceeding 350 pg/ml during the first 24hours following administration.

Initial burst can also be assessed by comparing circulatingconcentrations of the active pharmaceutical ingredient in a time periodimmediately following administration of the formulation with thecirculating concentration of the drug in a second time period thatimmediately follows the first. In one embodiment, use of theformulations of the present disclosure result in circulatingconcentrations of active pharmaceutical ingredient during the first 24hours following administration that do not exceed the circulatingconcentration during the next 24 hour period. In another embodiment, useof the formulations of the present disclosure result in averagecirculating concentration of active pharmaceutical ingredient during thefirst 24 hours following administration do not exceed the averagecirculating concentration during the next 24 hour period.

Methods of Storing

Another aspect provides methods of storing the sustained releaseformulations described herein. The methods of storing the formulationsdescribed herein may also be referred to as methods of preventing thedegradation of the microspheres. By “storing” is meant that theformulation is retained for a period of time within its containerwithout adding any additional component to the container and withoutremoving the formulation from the container (e.g., in the manufacturingfacility, during transport, in the pharmacy). The storage time willtypically be the amount of time between packaging of the formulation andits use by the patient. After the storage time the formulation isadministered to the patient in need thereof “Administering” to thepatient includes self-administration. The methods involve storing thesustained release formulations for a period of at least 1 week, at least2 weeks, at least 1 month, at least 3 months, at least 1 year, at least18 months, or at least 2 years. In some embodiments, the formulationscan be stored at 5° C. or 25° C. There is minimal degradation of themicrospheres when the formulations are stored for such extended periodsof time.

In another embodiment the invention provides methods of maintaining thepotency of (e.g., preventing the loss of biological activity) and/orpurity (e.g. preventing chemical changes in the molecule) an activepharmaceutical ingredient. Thus, a peptide or protein or other API thathas undergone a chemical change (e.g. oxidation) may result in a loss ofpurity, but may still retain its potency. The methods involve storing amicrosphere comprising a active pharmaceutical ingredient in anon-aqueous carrier as described herein for a period of time, wherebythe potency and/or purity of the active pharmaceutical ingredient ismaintained by the microspheres and the non-aqueous carrier. In theformulations described herein, at least 80%, at least 90%; at least 95%;at least 98%; or at least 99% of the potency and/or purity of the activepharmaceutical ingredient is retained for a period of time of at least 1week, at least 2 weeks, at least 1 month, at least 3 months, at least 1year, at least 18 months, or at least 2 years.

Methods of Administering/Treatment

In another aspect the present invention provides methods ofadministering an active pharmaceutical ingredient to a patient in needthereof. The methods involve administering to the patient a formulationor composition as described herein. Any of the formulations describedherein can be administered by parenteral administration, using any ofthe methods described herein. For example, the formulations can beadministered by subcutaneous, intra-muscular, intra-peritoneal,intra-abdominal, intravenous, or any suitable manner of administration.In one embodiment, the formulations described herein are administeredsubcutaneously. In one embodiment the methods involve injecting theformulation without the patient performing a prior step of combining thesustained release composition with a second carrier.

In one embodiment the administration does not comprise a mixing step. Amixing step is a step where the microspheres are combined with a carrierprior to injection. In various embodiments the mixing step is a stepwhere the microspheres are combined with a carrier within the 1 weekperiod prior to injection in the patient. The carrier can be anon-aqueous carrier, such as those described herein. Administration ofthe formulation refers to the complete process of the user interactingwith the formulation, including mixing, combining any ingredientsforming the formulation, and the actual injection or other form ofproviding the formulation to the patient.

The frequency of administration can vary depending on any one or acombination of factors such as the amount of the formulationadministered, the release profile of the formulation, the amount ofactive pharmaceutical ingredient in the formulation, and the circulatinglevel of active pharmaceutical ingredient to be achieved. In particularembodiments, the formulations described herein can be administered oncedaily, once per week, once every two weeks, once a month, once every twomonths, once every three months, once every four months, once every sixmonths or once per year. In one embodiment, the formulation isadministered once a week. In another embodiment, the formulation isadministered once a month.

When the formulations comprise a GLP-1 receptor agonist, such as GLP-1or an analog thereof, or an exendin (e.g., exenatide) or an analogthereof, they can be used to treat numerous diseases, such as diabetes(e.g., Type 1 diabetes, Type II diabetes, gestational diabetes),impaired glucose tolerance, hyperglycemia (e.g., fasting andpostprandial), obesity, overweight, non-alcoholic fatty liver disease,non-alcoholic steatohepatitis (NASH), and the like. The formulationscomprising a GLP-1 receptor agonist (e.g., exenatide) will also beuseful to stimulate insulin release; lower plasma glucagon; reduce foodintake, reduce appetite, decrease gastric motility, delay gastricemptying, lower plasma lipid (e.g., triglycerides, cholesterol) levels,and the like. These methods of treatment are described, for example, inU.S. Pat. Nos. 5,424,286, 6,858,576, 6,872,700, 6,956,025, and6,956,025, and WO 2007/022518, the disclosures of which are incorporatedby reference herein.

In certain embodiments, administration of any of the formulationsprovided herein comprising a glucoregulatory peptide such as an exendin,e.g. exenatide, result in a 2 hour plasma glucose of less than 300mg/dl, less than 275 mg/dl, less than 250 mg/dl, or less than 225 mg/dl.In a particular embodiment administration of any of the formulationsprovided herein comprising a glucoregulatory peptide such as an exendin,e.g. exenatide, results in a 2 hour plasma glucose of less than 200mg/dl. In other embodiments, administration of any of the formulationsprovided herein comprising a glucoregulatory peptide such as an exendin,e.g. exenatide, results in a 2 hour plasma glucose of less than 190mg/dl, less than 180 mg/dl, less than 170 mg/dl, less than 160 mg/dl, orless than 150 mg/dl. In certain embodiments, administration of any ofthe formulations provided herein comprising a glucoregulatory peptidesuch as an exendin, e.g. exenatide, results in a 2 hour plasma glucoseless than 140 mg/dl. In further embodiments, administration of any ofthe formulations provided herein comprising a glucoregulatory peptidesuch as an exendin, e.g. exenatide, results in a venous or capillaryfasting blood glucose (FBG) level of less than 200 mg/dl, less than 175mg/dl, less than 150 mg/dl, less than 140 mg/dl, less than 130 mg/dl,less than 120 mg/dl, or less than 115 mg/dl. In one embodiment, a FBGlevel of less than 110 mg/dl is achieved, while in another embodiment aFBG level of less than 100 mg/dl is achieved.

In additional embodiments, administration of any of the formulationsprovided herein comprising a glucoregulatory peptide such as an exendin,e.g. exenatide, results in a 2 hour venous or capillary blood glucoselevel of less than 300 mg/dl, less than 275 mg/dl, less than 250 mg/dl,less than 225 mg/dl, or less than 200 mg/dl. In a particular embodimentadministration of any of the formulations provided herein comprising aglucoregulatory peptide such as an exendin, e.g. exenatide, results in a2 hour blood glucose level of less than 180 mg/dl. In furtherembodiments, administration of any of the formulations provided hereincomprising a glucoregulatory peptide such as an exendin, e.g. exenatide,result in blood glucose levels of less than 170 mg/dl, less than 160mg/dl, less than 150 mg/dl, less than 140 mg/dl, less than 130 mg/dl, orless than 120 mg/dl. In particular embodiments, administration of any ofthe formulations provided herein comprising a glucoregulatory peptidesuch as an exendin, e.g. exenatide, result in a 2 hour venous bloodglucose level of less than 120 mg/dl, while in other embodiments, a 2hour capillary blood glucose level of less than 140 mg/dl is achieved.

In one embodiment, glucose levels are average glucose levels calculatedover a chosen time period. Specific examples include, but are notlimited to, daily average glucose levels, weekly average glucose levels,monthly average glucose levels or yearly average glucose levels. Twohour circulating glucose levels are determined after an oral glucosetolerance test (OGTT). In the standard test, 75 g of anhydrous glucoseis dissolved in 250-300 ml of water and administered over 5 minutes. Inchildren, glucose is administered at a rate of 1.75 g/kg body weight upto a maximum of 75 grams of glucose. A baseline glucose level isobtained prior to ingestion and then typically every 30 minutes for 2hours. For gestational diabetes, a 100 g, 3 hour test is often used.

Because glucose freely crosses the cell membrane of red blood cells,erythrocyte hemoglobin undergoes a nonenzymatic glycosylation at theamine residues. Hemoglobin A1c (HbAlc) refers to the percentage ofhemoglobin molecules with glucose moieties attached to the N-terminalvalines of each of the two beta-chains. Glycohemoglobin includes HbAlcalong with other forms of hemoglobin where glycosylation has occurred atother amino acids. The percentage of hemoglobin molecules undergoingglycosylation is proportional to the average ambient glucoseconcentrations during the previous during the previous 60-90 days. HbAlcis a commonly used measure to assess the state of glycemic control inpatients with diabetes.

In one embodiment, administration of any of the formulations providedherein comprising a glucoregulatory peptide such as an exendin, e.g.exenatide, results in a reduction to, maintenance of, or both of HbAlclevels of less than 8%. In another embodiment HbAlc levels are reducedto, maintained at, or both to less than 7.5%, while in yet anotherembodiment, HbAlc levels are reduced to, maintained at, or both at lessthan 7%. In further embodiments, administration of any of theformulations provided herein comprising a glucoregulatory peptide suchas an exendin, e.g. exenatide, results in a reduction to or maintenanceof, or both of HbAlc levels to less than 6.5%, less than 6%, less than5.5%, less than 5% less than 4.5% or less than 4%. Thus, thecompositions disclosed herein are useful in a method of reducing ormaintaining HbAlc levels in the blood, the methods comprisingadministering a composition disclosed herein. In another embodiment,administration of any of the formulations provided herein comprising aglucoregulatory peptide such as an exendin, e.g. exenatide, results in areduction to, maintenance of, or both of glycosylated hemoglobin levelsof less than 10%. In another embodiment, glycosylated hemoglobin levelsare reduced to, maintained at, or both to less than 9.5%; while in yetanother embodiment, glycosylated hemoglobin levels are reduced to,maintained at, or both at less than 9%. In further embodimentsadministration of any of the formulations provided herein comprising aglucoregulatory peptide such as an exendin, e.g. exenatide, results in areduction to, or maintenance of, or both of glycosylated hemoglobinlevels to less than 8.5%, less than 8%, less than 7.5%, less than 7%less than 6.5%, less than 6%, less than 5.5%, less than 5%, less than4.5% or less than 4%. In other aspects administration of any of theformulations provided herein comprising a glucoregulatory peptide suchas an exendin, e.g. exenatide, results in a lower of HbAlc by at least0.2%, at least 0.4%, at least 0.6%, at least 0.8%, at least 1%, at least1.2%, at least 1.4%, at least 1.6%, at least 1.8%, or at least 2%. Thus,the invention provides methods of reducing or maintaining glycosylatedhemoglobin levels in the blood, the methods involving administering acomposition described herein.

It should be realized that a subject in need of lowering of bloodglucose is not limited to patients having diabetes mellitus, but mayinclude any subject suffering from hyperglycemia for whatever reasonincluding, but not limited to, injury, trauma, surgery, stroke andmyocardial infarction. The amount of glucose lowering will vary with thesubject in question and depend on factors such as the severity of thehyperglycemia and the severity of the disease, disorder or condition inquestion.

EXAMPLES

The following non-limiting examples provide further illustrations ofmaking and using the formulations described herein, and are not intendedto limit the scope of the appended claims. With respect to the Examplesherein, MCT oil refers to medium chain triglyceride oil which iscommercially available as MIGLYOL® 812 (Sasol Germany GmbH, Witten,Germany).

Example 1

Microspheres may be prepared by processes known in the art anddescribed, e.g., in U.S. Pat. Nos. 7,563,871 and 7,456,254. Microspherescomprising a poly(lactide-co-glycolide) copolymer having dispersedtherein 5% (w/w) exenatide and 2% (w/w) sucrose were obtained. Thepoly(lactide-co-glycolide) copolymer had a ratio of lactide:glycolide of1:1. These microspheres are currently being developed by AmylinPharmaceuticals, Inc. (San Diego, Calif.), Alkermes, Inc. (Cambridge,Mass.), and Eli Lilly and Company (Indianapolis, Ind.) for a once-weeklyformulation for treating diabetes. Gedulin et al, Diabetologia,48:1380-1385 (2004).

Example 2

The stability of the microspheres from Example 1 was investigated todetermine their stability over an extended period of time while storedin a non-aqueous carrier. Microspheres from Example 1 were stored for aperiod of 6 months at 5° C. in a formulation comprising a non-aqueouscarrier (i.e., sesame oil; MCT oil; and ethyl oleate, which is amonoglyceride). The control was an aqueous formulation comprising themicrospheres from Example 1 in an aqueous carrier containingcarboxymethylcellulose and a surfactant.

The stability of the microspheres was determined by morphology andparticle size via examination under a microscope. Exenatide purity,potency (by HPLC evaluation), and in vitro release were also determined.As shown in Table 1, after 6 months of storage the physical structure(i.e., size, morphology) of the microspheres did not change.

As shown in Table 2, the microspheres stored in a MCT oil showed nochange in the purity of exenatide based on an HPLC analysis. Impuritiesmight also be referred to as degradation products from the peptide. Highpurity means relatively little degradation of the peptide. The purity isrelative to the formulation at time zero. The microspheres stored insesame oil and ethyl oleate showed a slight decrease in the purity ofexenatide. The impurities did not appear to be oil orpoly(lactide-co-glycolide) polymer related (based on retention times),but appeared to be related to the stability of exenatide itself.

Table 3 shows that the potency of exenatide did not significantlydecrease over the 6 month period regardless of the non-aqueous carrierthat was used.

TABLE 1 Particle size and morphology using microscope size (μm)(standard deviation (μm)) morphology T = 0 1 month 6 months 0 to 6months sesame oil 64 (22) 63 (23) 64 (12) no change MCT oil 65 (19) 60(22) 61 (17) no change ethyl oleate 64 (16) 62 (16) 59 (13) no change

TABLE 2 Change in Purity of Exenatide Containing Formulation % purity ofexenatide 1 % 3 % 6 % t = 0 month change* month change* month change*sesame 95.93 95.68 −0.25 94.55 −1.38 95.00 −0.93 oil MCT 95.63 95.56−0.07 94.67 −0.96 95.50 −0.13 oil ethyl 95.60 95.80 0.20 93.67 −1.9394.70 −0.90 oleate *Changes less than 0.5% are considered to beinsignificant

TABLE 3 Change in Potency of Exenatide Based on Carrier in Formulationcarrier time zero 1 month 3 months 6 months sesame oil 97 104 98 98 MCToil 94 108 99 99 ethyl oleate 95 98 99 100

Example 3

The pharmacokinetics of the formulations in Example 2 were determined,except that 2% (w/w) lecithin was added to the ethyl oleate carrier.Single injections with a dose of 53 mg/ml of microspheres per ml ofnon-aqueous carrier were administered to 6 rats with a 21 G needle. Inthe study, a comparison was also made to the microspheres from Example 1that were mixed with an aqueous carrier just before injection.

FIG. 1 provides a comparison of the pharmacokinetics of the fourdifferent formulations of microspheres containing exenatide. In threeformulations, the carrier is an oil (e.g., sesame oil; MCT oil; ethyloleate). In one comparative formulation, the carrier is an aqueousdiluent. As can be seen from the data, the formulations having an oilcarrier had reduced burst when compared to the formulation having anaqueous carrier.

FIG. 2 is a graphical simulation of data extrapolated from FIG. 1 of theplasma exenatide concentration over time of the formulation comprisingthe MCT oil carrier and the comparative formulation comprising theaqueous carrier. The plasma concentration plateau of exenatide may bereached after about 5 dosings.

Example 4

A formulation comprising the microspheres of Example 1 in an aqueouscarrier and a formulation comprising the microspheres of Example 1 in anMCT carrier were prepared. The burst release was evaluated by addingabout 0.75 mL of the formulations to a 10 mM HEPES release buffer. Themixture was agitated to ensure that the microspheres achieved fullcontact with the HEPES release buffer. After incubation at 37° C. forone hour, the mixture was centrifuged and the aqueous phase was analyzedby HPLC to determine the burst release. The concentration of the dosetested for release was 150 mg/mL.

FIG. 3 shows the lower burst release of the formulation having the oilcarrier compared to the formulations having an aqueous carrier. Thegraph shows that with an aqueous carrier, about 0.6% of exenatide wasreleased in the burst. With the formulation having the MCT oil carrier,less than 0.1% of exenatide was released in the burst.

FIG. 4 illustrates the in vivo release profile in rats over 10 hours forthe formulation of Example 1 in MCT oil compared to a formulationcomprising the same microspheres in an aqueous (saline) carrier. In thetime period following sub-cutaneous administration of the formulation,the entrance of exenatide into the plasma was markedly lower than thesame microspheres administered in the aqueous carrier. The formulationof the invention shows no burst release, and a markedly more gradualentrance into the blood plasma versus the aqueous formulation. Incontrast, the aqueous formulation showed a burst release followed by asharper entrance into the blood plasma.

Example 5

Microparticles were prepared in a manner similar to that described inthe examples in U.S. Pat. No. 5,439,688, the disclosure of which isincorporated by reference herein. Eight samples were prepared by brieflymixing an active pharmaceutical ingredient (i.e., davalintide,pramlintide, metreleptin, bovine serum albumin, sodium salicylate,salicylic acid, minocycline HCl, insulin) and polymer (i.e,poly(lactide-co-glycolide) copolymer or polycaprolactone/PLGA copolymer)and then the mixture was placed in a grinder to obtain awell-homogenized powder. Mixtures ranged from 2% to 10% w/w of theactive pharmaceutical ingredient. The mixed powder was transferred to anextruder where the temperature was adjusted according to the chosenpolymer. Some polymers needed higher temperatures to produce a melt withgood flow properties. The extruder contained twin screws that movedclockwise to produce efficient mixing. The material was extruded througha 1.5 mm orifice, collected, cooled at room temperature, and cut intoshort strands about 1-2 inches long. These strands were then fed into a12-tooth rotor mill, followed by a sieving step to producemicroparticles of about 20 to 100 microns. The microparticles werecollected and stored at 5° C. until further use.

Experimental samples were prepared by dispersing about 50 mg of themicroparticles into 0.75 mL of a MCT oil carrier. The samples werestored at 5° C. and 25° C. for two days, two weeks, or one month, atwhich times representative samples were tested. The fraction of drugthat remained in the microparticles and the fraction of drug thatpartitioned into the MCT oil carrier were determined. Briefly, thesamples were centrifuged to separate the microparticles from the MCT oilcarrier. Each portion was treated independently to determine the amountof drug it contained. Results are reported on the basis of the percentresiding in each independent portion.

TABLE 4 PLGA copolymer; 2 Days Storage at 5° C. Compound MicroparticlesMCT Carrier davalintide 99.8% 0.2% pramlintide 100.0% 0.0% metreleptin100.0% 0.0% bovine serum albumin 100.0% 0.0% sodium salicylate 99.5%0.5% salicylic acid 98.9% 1.1% minocycline 99.1% 0.9%

TABLE 5 PLGA copolymer; 1 Month Storage at 5° C. Compound MicroparticlesMCT Carrier davalintide 99.4% 0.6% pramlintide 99.7% 0.3% metreleptin100.0% 0.0% bovine serum albumin 100.0% 0.0% sodium salicylate 98.7%1.3% salicylic acid 99.9% 0.1% minocycline 99.9% 0.1% insulin 99.5% 0.5%

TABLE 6 PLGA copolymer; 2 Days Storage at 25° C. Compound MicroparticlesMCT Carrier davalintide 100.0% 0.0% pramlintide 100.0% 0.0% metreleptin100.0% 0.0% bovine serum albumin 100.0% 0.0% sodium salicylate 97.7%2.3% salicylic acid 99.1% 0.9% minocycline 99.4% 0.6%

TABLE 7 PLGA copolymer; 1 Month Storage at 25° C. PLGA Polymer; 1 MonthStorage at 25° C. Compound Microparticles MCT Carrier davalintide 100.0%0.0% pramlintide 100.0% 0.0% metreleptin 100.0% 0.0% bovine serumalbumin 100.0% 0.0% sodium salicylate 98.5% 1.5% salicylic acid 99.8%0.2% minocycline 99.6% 0.4% insulin 99.3% 0.7%

TABLE 8 polycaprolactone/PLGA copolymer; Two Weeks Storage 5° C. 25° C.MCT MCT Compound Microparticles Carrier Microparticles Carrierpramlintide 100.0% 0.0% 100.0% 0.0%

The data in Tables 4-8 illustrate the broad applicability of thesustained release formulations described herein to a variety ofdifferent active pharmaceutical ingredients, including peptides andsmall molecules. The compositions have been successfully produced usinga variety of peptides, bovine serum albumin, and even a selection ofsmall molecules. Surprisingly salicylic acid, which is oil soluble, didnot migrate into the MCT carrier oil, despite that its solubility in theMCT oil is greater than 30 mg/ml. Thus, the microparticles remain intactupon storage in MCT even when the active pharmaceutical ingredient issoluble in MCT. The data further illustrate that the compositions can besuccessfully produced even using other polymer mixtures in themicroparticles.

Example 6

The percentage purity of exenatide was measured by HPLC at one monthintervals over a 9 month period in the following four formulations: (i)a formulation comprising the microspheres of Example 1 stored in an oilMCT oil carrier at 5° C.; (ii) a formulation comprising the microspheresof Example 1 stored in an MCT oil carrier at 25° C.; (iii) drymicrospheres of Example 1 that had been stored in a container for 9months at 5° C. without a liquid carrier, and that were then admixedwith an aqueous carrier immediately prior to the study; and (iv) drymicrospheres of Example 1 that had been stored in a container for 9months at 25° C. without a liquid carrier, and that were then admixedwith an aqueous carrier immediately prior to the study.

FIGS. 5A and B show the following: (i) exenatide had a purity greaterthan 93% at 6 months and 9 months in the formulation with the oilcarrier at a temperature of 5° C.; (ii) exenatide had a purity greaterthan 86% at 6 months and 9 months in the formulation with the oilcarrier at a temperature of 25° C.; (iii) exenatide had a purity ofgreater than 94% at 6 months where the microspheres had been stored dryat 5° C.; and (iv) exenatide had a purity of greater than 90% at 6months in the formulation where the microspheres had been stored dry ata temperature of 25° C. In FIG. 5A, the purity of exenatide wasdetermined by strong cation exchange HPLC. In FIG. 5B, the purity ofexenatide was determined by reverse-phase HPLC.

Example 7

Formulations containing the microspheres from Example 1 and an MCT oilcarrier were stored at 5° and the potency of exenatide was measured atmonthly intervals for 9 months. Additionally, formulations containingthe microspheres from Example 1 and an MCT oil carrier were stored at25° and the potency of exenatide was measured at monthly intervals for 6months. FIG. 6 presents the results which show that the potency ofexenatide was preserved for at least 9 months.

Example 8

The physical integrity of a formulation containing the microspheres fromExample 1 in an MCT oil carrier was analyzed. After storage for a periodof 6 months at 5° C., the molecular weight of thepoly(lactide-co-glycolide) copolymer did not change relative to timezero. After storage for a period of 6 months at 25° C., the molecularweight of the poly(lactide-co-glycolide) copolymer decreased by 6kDaltons, which was comparable to the molecular weight change of drymicrospheres (i.e., microspheres stored for 6 months at 25° C. not inany carrier). The mean diameter of the microspheres was measured afterstorage at 3, 6, and 9 months at either 5° C. or 25° C., and no changein mean diameter was detected relative to time zero.

Example 9

The ratio of lactide/glycolide for the microparticles was alsoinvestigated for use with various APIs. The Table below provides thevarious lactide/glycolide ratios used.

Approx polymer MW Lactide/Glycolide Polymer Drug (kDa) ratio for PLGAPLGA davalintide 10 50/50 PLGA pramlintide 10 50/50 PLGA Leptin 10 75/25PLGA BSA 25 50/50 PLGA Na Salicylate 25 50/50 PLGA Salicylic acid 2550/50 PLGA Minocycline 10 75/25 PLGA Insulin 25 50/50 1.1:1 PCL/PLGApramlintide PCL = 150 50/50 PLGA = 10

All publications and patents are incorporated by reference herein. Theforegoing has been described in detail, and the skilled artisan willrecognize that modifications may be made without departing from thespirit or scope of the disclosure or appended claims.

1. A manufactured pre-mixed formulation for injection comprising asuspension of: a pharmaceutically acceptable non-aqueous carriercomprising one or more triglycerides of C₆-C₁₂ fatty acids; andmicrospheres comprising a poly(lactide-co-glycolide) copolymer andexenatide; wherein the microspheres have a mean diameter of volumedistribution of about 50 microns to 70 microns, and wherein themicrospheres have a non-porous outer layer.
 2. (canceled)
 3. Themanufactured pre-mixed formulation of claim 1, wherein the carrier isnot contained within the interior spaces or pores of the microspheres.4. The manufactured pre-mixed formulation of claim 1, wherein themicrospheres further comprise a sugar.
 5. The manufactured pre-mixedformulation of claim 4, wherein the sugar is selected from the groupconsisting of glucose, dextrose, galactose, maltose, fructose, mannose,sucrose, lactose, trehalose, raffinose, acarbose, glycol, glycerol,erythritol, threitol, arabitol, ribitol, sorbitol, dulcitol, iditol,isomalt, maltitol, lactitol, mannitol, xylitol, and a combinationthereof.
 6. The manufactured pre-mixed formulation of claim 5, whereinthe sugar is sucrose.
 7. The manufactured pre-mixed formulation of claim1, wherein the biocompatible, biodegradable polymer is selected from thegroup consisting of a polylactide, a copolymer of a polylactide, apolyglycolide, a copolymer of a polyglycolide, apoly(lactide-co-glycolide) copolymer, a polylactic acid, a copolymer ofa polylactic acid, a polyglycolic acid, a copolymer of a polyglycolicacid, a poly(lactic acid-co-glycolic acid) copolymer, apolycaprolactone, a copolymer of a polycaprolactone, a polycarbonate, acopolymer of a polycarbonate, a polyesteramide, a copolymer of apolyesteramide, a polyanhydride, a copolymer of a polyanhydride, apolyamino acid, a copolymer of a polyamino acid, a polyorthoester, acopolymer of a polyorthoester, a polycyanoacrylate, a copolymer of apolycyanoacrylate, a poly(p-dioxanone), a copolymer of apoly(p-dioxanone), a polyalkylene oxalate, a copolymer of a polyalkyleneoxalate, a polyurethane, a copolymer of a polyurethane, and acombination thereof.
 8. (canceled)
 9. The manufactured pre-mixedformulation of claim 1, further comprising a pharmaceutically acceptableexcipient.
 10. The manufactured pre-mixed formulation of claim 9,wherein the pharmaceutically acceptable excipient is selected from thegroup consisting of a sugar, a sugar alcohol, an antioxidant, apreservative, and a combination thereof.
 11. The manufactured pre-mixedformulation of claim 9, wherein the pharmaceutically acceptableexcipient is selected from the group consisting of sucrose, glucose,dextrose, galactose, maltose, trehalose, fructose, maltodextrin, glycol,glycerol, erythritol, threitol, arabitol, ribitol, sorbitol, dulcitol,iditol, isomalt, maltitol, lactitol, mannitol, xylitol, benzoic acid,sorbic acid, meta cresol, sodium benzoate, potassium sorbate,methylparaben, propylparaben, butylparaben, benzalkonium chloride,sodium metabisulfite, butylated hydroxy anisole, butylated hydroxytoluene, sodium sulfite, tocopherol thymol, ascorbate, propyl gallate,and a combination thereof.
 12. The manufactured pre-mixed formulation ofclaim 9, wherein the pharmaceutically acceptable excipient is sucrose.13. (canceled)
 14. The manufactured pre-mixed formulation of claim 4,wherein the microspheres comprise a poly(lactide-co-glycolide) copolymeras the biocompatible, biodegradable polymer; wherein thepoly(lactide-co-glycolide) copolymer has dispersed therein 1% to 10%(w/w) exenatide and 0.1% to 5% (w/w) sugar.
 15. The manufacturedpre-mixed formulation of claim 7, wherein the microspheres comprise apoly(lactide-co-glycolide) copolymer as the biocompatible, biodegradablepolymer; wherein the poly(lactide-co-glycolide) copolymer has dispersedtherein 5% (w/w) exenatide and 2% (w/w) sucrose.
 16. The manufacturedpre-mixed formulation of claim 1, wherein the pharmaceuticallyacceptable non-aqueous carrier further comprises up to 2% of C₁₄ fattyacids.
 17. The manufactured pre-mixed formulation of claim 1, whereinthe microspheres are present in the formulation at a concentration offrom 10 mg/ml to 500 mg/ml or from 20 mg/ml to 200 mg/ml.
 18. A methodfor treating diabetes, stimulating insulin release; lowering plasmaglucagon; reducing food intake; reducing appetite; decreasing gastricmotility; delaying gastric emptying; lowering plasma lipid levels;treating impaired glucose tolerance; treating hyperglycemia; treatingobesity; treating overweight; treating fatty liver disease; or treatingnon-alcoholic steatohepatitis in a patient in need thereof, comprisingadministering to the patient the manufactured pre-mixed formulation ofclaim
 1. 19. A kit comprising a container which comprises themanufactured pre-mixed formulation of claim 1 and instructions for use;wherein the container is selected from the group consisting of asingle-dose pen injector, a multi-dose pen injector, a single-dose vial,a multi-dose vial, a single-dose cartridge and a multi-dose cartridge.20. The kit of claim 19, wherein the container is a single-dose peninjector.